Pronase

Immunofluorescence staining of pancreas cryosections was performed as described [22 (link)]. Sections were fixed in methanol at −20°C, washed, blocked with 1% BSA in PBS and incubated overnight at 4°C with primary antibody diluted in blocking solution. After washing, sections were incubated overnight at 4°C with secondary antibodies. Paraffin sections were rehydrated and submitted to antigen retrieval by heating the sections for 30 min in 10 mmol/l citrate buffer, pH 6.0 in a microwave (700 W). Sections were treated with Pronase (Roche, Germany) (1 mg/ml Pronase in 50 mmol/l Tris-HCl [pH 7.5] and 5 mmol/l EDTA) for 15 min at 37°C to retrieve masked ECM molecules. Blocking and incubation with the primary and secondary antibodies were performed as for cryosections. The primary antibodies employed are listed in electronic supplementary material (ESM) Table 1. Human thymus and LN sections were used to validate the primary antibodies. The specificity of secondary antibodies was verified by omitting the primary antibodies from the staining procedure (ESM Fig. 1). The sections were examined using a Zeiss AxioImager (Zeiss, Germany) or an LSM700 microscope (Zeiss).

Cell lysates were obtained from the KYSE30 cells. Cells were scraped and lysed with HEPES lysis buffer containing a protease inhibitor and a phosphatase inhibitor. After centrifugation at 12,000 × g for 15 min, the protein concentration was quantified using the BCA Protein Assay Kit (Beyotime, China). Before drug treatment, the protein concentration was diluted to 5 mg/mL. Then, the cell lysates were treated with the verdinexor and DMSO for 3 h at 37 ℃. Samples were incubated with pronase (Roche, Switzerland, 1 μg pronase with 2 mg proteins, 1:2000) or distilled water for 20 min at 37 ℃. After protease digestion, the candidate target proteins were detected by western blot analysis. Β-Actin was used as an internal control.

Hepatocyte isolation was achieved through 2-step perfusion of the liver in situ using Liver Perfusion and Liver Digest Media (Thermo Fisher Scientific) and then density separation by 50% Percoll (GE Healthcare Life Sciences) gradient. Stellate cell isolation was performed following previously published protocols (82 (link)). Briefly, fibrotic livers were perfused in situ with 1.2 mg/mL pronase (Roche) and 0.25 U/mL collagenase (Crescent Chemical) and then digested in vitro with 0.5 mg/mL pronase, 0.1 U/mL collagenase, and 0.1 mg/mL DNase I (Roche) for 10 minutes in a 250 rpm shaker at 37°C. Stellate cells were separated by density gradient centrifugation using 12% Accudenz (Accurate Chemical) at 1380g for 17 minutes at 4°C.

The DARTS assay to test the binding of SA to PP2AA1-GFP was performed as previously reported [77 (link), 78 (link)]. pPP2AA1::PP2AA1-GFP seedlings (7d) were used for total protein extraction. After harvesting, the samples were ground in liquid nitrogen, resuspended in protein extraction buffer (25 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% IGEPAL CA-630, Roche cOmplete protease inhibitor cocktail, EDTA free) with a 1:2 (w/v) ratio, and spun down to discard the cell debris. After quantifying the protein concentration (Quick Start Bradford Reagent, Bio-Rad), the cell lysate was aliquoted and incubated with 0, 50 μM or 500 μM SA respectively for 30 min at 25°C, mixing at a low speed. The treated extracts were further aliquoted, and mixed with different concentrations of Pronase (Roche) in Pronase buffer (25 mM Tris-HCl, pH 7.5; 150 mM NaCl). After incubation at 25°C for 30 min, the proteolytic digestion was terminated by adding protease inhibitor cocktail (cOmplete, Roche) and the samples were kept on ice for 10 min. The protein samples were then analyzed by western blot. PP2AA1-GFP was detected by an anti-GFP antibody (JL8, Clontech, 1:2000). HRP activity was detected by the Supersignal Western Detection Reagents (Thermo Scientific) and imaged with a GE Healthcare Amersham 600RGB system.